Spontaneous mutations contribute significantly to human genetic disease and form the background against which the genetic toxicity of environmental agents must be judged. In spite of these central concerns, the cellular processes that influence the spontaneous mutation rate are poorly understood. This project addresses key issues regarding the biochemical mechanisms of spontaneous mutagenesis eukaryotes: Is spontaneous mutation affected by processing of a gene by transcription and replication (Aim 1)? Is spontaneous mutation driven by metabolically generated DNA damage such as oxygen radicals (Aims 2-4)? Do repairable spontaneous DNA damages escape correction and cause mutations in normal cells (Aim 3)? Is their probability of generating mutations more a question of kinetics (repair vs. replication) or the rate at which damage is generated (Aim 4)? Do mutations due to endogenous DNA damage result from active processing pathways that handle environmental mutagens (Aim 5)? To achieve these ends, we will exploit our ability to modulate the DNA repair capacity of the yeast Saccharomyces cerevisiae by inactivating the APN1 gene we cloned previously, which encodes the main endonuclease for abasic sites in this organism. Strains lacking this activity have a substantially elevated rate of spontaneous mutation, which is generated in part by abasic sites produced by a DNA glycosylase (the MAG gene product) acting on endogenous DNA damages. We will construct test strains bearing a mutation targets of human origin, in common with the other projects. Rapid determination of mutation spectra using a newly-developed approach will allow us to tease out specific events that limit normal genetic stability. These targets will be inserted with different orientations respective to replication and transcription (leading vs. lagging strand, and transcribed vs. nontranscribed strand). The role of endogenous damage will be assessed by deleting the APN1 or MAG genes, and by culturing the cells under different conditions (aerobic vs. anaerobic, etc.). Yeast strains will also be constructed with increased levels of Apn1 protein or the human abasic endonuclease, Ape, which will reveal whether repair of such damages is limiting in normal cells. Finally, mutations in the RAD6 or REV3 genes will be introduced to reveal whether active mutational systems contribute to "unprogrammed" genetic change.